The goal of this proposal is to identify molecular interactions relevant to drug targeting and chemical signaling in cell membranes. More than half the drugs on the market target membrane proteins, a class of molecules where obtaining molecular detail in intact membranes challenges existing methods. We will use the plasmonic properties of nanoparticles to enable chemical-specific spectroscopic studies of ligand-receptor binding in intact cellular membranes. These investigations will demonstrate a new approach to probe the receptor's chemical residues that bind peptide antagonists and provide insight into molecular interactions that regulate the membrane proteins involved in signaling and drug targeting. Our approach combines tip enhanced Raman scattering (TERS), surface plasmon resonance (SPR) spectroscopy, and single particle tracking to characterize chemical interactions that regulate binding to membrane receptors. TERS will determine the chemical residues present in cell membranes that are associated with ligand binding. Initial results obtained in our lab indicate that ligand-functionalized nanoparticles selectively enhance the spectroscopic signals of the receptor involved in ligand binding. The ligand affinity can be determined from proteins immobilized on surfaces using SPR measurements. Complementary measurements can be performed in cells using single particle tracking. By taking advantage of technology that enables tracking of nanoparticles in real-time, we will characterize both ligand binding and membrane organization of individual ligand-functionalized nanoparticles bound to receptors on living cells. The specific aims of this proposal are: 1) Correlate in situ studies ligand affinity with the molecular identity of the receptor associated with peptide binding to the integrin receptors by combing TERS and SPR. 2) Explore protein receptor-binding interactions in vitro between integrin receptors and peptide ligands using targeted TERS. 3) Correlate in vitro binding affinity with chemical interaction by combining single nanoparticle tracking studies with TERS studies. The technology and platform we propose will address the challenge of obtaining chemical information from ligands binding to receptor proteins in intact cell membranes. These studies will provide new insights into the molecular interactions that regulate signaling pathways and how anomalies in these interactions are associated with disease and treatment.